Supplementary Information

A Holistic View into the Gallic Acid-induced Attenuation in Colitis

Based on the Microbiome-Metabolomics Analysis

Yuan Lia†, Zhiyong Xieb, c†, Tingting Gaoa, Lin Lia, Yongda Chena, Dan Xiaob, Wen

Liub, Baorong Zoub, Biyu Lua, Xing Tiand, Bo Hand, Yaping Guoe, Shaobao Zhangb,

Lei Linb, Mengxia Wanga, Pei Lia, Qiongfeng Liaoa*

a. School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine,

Guangzhou, China.

b. School of Pharmaceutical Sciences (Shenzhen), Sun Yat-sen University,

Guangzhou, 510006, China

c. Guangdong Provincial Key Laboratory of New Drug Design and Evaluation,

Guangzhou, China

d. School of Pharmacy, Xinjiang Shihezi University, Xinjiang, 832002, China

e. The first affiliated hospital, School of medicine, Xinjiang Shihezi university,

Xinjiang, 832002, China.

† These authors contributed equally to this work and should be considered co-first

authors.

*Corresponding authors:

Qiongfeng Liao

Tel./Fax: +86 20 3935 8081E-mail: liaoqf2075@yahoo.com

Electronic Supplementary Material (ESI) for Food & Function.This journal is © The Royal Society of Chemistry 2019

Figure 1. LDA score plot

Figure 2. PCA score plot based on PICRUSt analysis

Figure 3. PCA scores plots of 1H NMR data of feces and urine

Table 1. Relative abundance of major bacterial taxa among different groups

Table 2. 1H NMR data of metabolites in rat feces

Table 3. 1H NMR data of metabolites in rat urine

Table 4. Screened metabolites with significant difference in fold change (FC)

value in feces samples

Table 5. Screened metabolites with significant difference in fold change (FC)

value in urine samples

Figure 1. LDA score plot

Figure 2. PCA score plot based on PICRUSt analysis

PICRUSt analysis shows the relative abundance of predicted microbial genes related to metabolism among groups based on Welch’s t test.

Figure 3. PCA scores plots of 1H NMR data of feces and urine

Table 1. Relative abundance of major bacterial taxa among different groupsPhylum Family Genus Relative abundance(%)

Control DSS DSS±GACorynebacteriaceae Corynebacterium 0.19±0.17 0.00±0.00## 0.04±0.05

Micrococcaceae Rothia 0.01±0.00 0.01±0.01 0.01±0.01Bifidobacteriaceae Bifidobacterium 0.03±0.06 0.02±0.05 0.03±0.06Actinobacteria

Coriobacteriaceae Adlercreutzia 0.01±0.00 0.00±.0.00## 0.01±0.01

Bacteroidaceae Bacteroides 0.77±0.36 5.54±4.57## 19.70±9.77*##

Porphyromonadaceae Parabacteroides 0.19±0.07 2.04±2.54## 1.33±1.39#

Prevotellaceae Prevotella 31.82±9.06 3.33±5.58## 0.00±0.00*##

Rikenellaceae AF12 0.05±0.04 0.00±0.00## 0.00±0.00##

S24-7 g_ 25.84±3.54 21.2±3.13 25.31±13.00Butyricimonas 0.03±0.03 0.04±0.04 0.00±0.00*##

[Odoribacteraceae]Odoribacter 0.01±0.01 0.01±0.01 0.00±0.00

[Paraprevotellaceae] Paraprevotella 4.84±5.27 5.39±5.38 3.03±1.42

Bacteroidetes

[Paraprevotellaceae] [Prevotella] 2.23±2.12 7.66±2.46## 9.68±8.20Deferribacteres Deferribacteraceae Mucispirillum 0.01±0.01 0.01±0.01 0.00±0.00

Lactobacillaceae Lactobacillus 4.40±2.02 0.61±0.49## 2.10±1.78

Streptococcaceae Lactococcus 0.00±0.00 0.02±0.02## 0.00±0.00**

Turicibacteraceae Turicibacter 0.06±0.10 0.69±0.63## 0.13±0.13*

Candidatus Arthromitus 0.00±0.00 0.00±0.00 0.01±0.01Clostridiaceae Clostridium 0.08±0.07 0.26±0.31 0.04±0.06Dehalobacteriaceae Dehalobacterium 0.04±0.02 0.02±0.02 0.10.±0.08

Blautia 0.05±0.11 0.26±.32 0.10±0.14Coprococcus 0.20±0.32 0.35±0.16 0.57±0.44

Dorea 0.04±0.02 0.20±0.19## 0.10±0.13

Firmicutes

Lachnospiraceae

Lachnospira 0.01±0.01 0.00±0.00 0.04±0.07

Roseburia 0.03±0.03 0.09±0.22 0.10±0.17[Ruminococcus] 0.19±0.15 0.35±0.47 0.13±0.08

Peptococcaceae rc4-4 0.02±0.02 0.13±0.03## 0.37±0.25*##

Oscillospira 2.33±1.10 5.67±4.32 5.01±2.89Ruminococcaceae Ruminococcus 4.59±1.34 2.75±0.85## 3.50±1.63

Veillonellaceae Phascolarctobacterium 0.30±0.72 1.35±0.80# 0.98±1.39

Allobaculum 0.01±0.00 0.44±0.35## 0.22±0.44##Erysipelotrichaceae

p-75-a5 0.01±0.01 0.01±0.01 0.00±0.00Alcaligenaceae Sutterella 0.42±0.17 2.70±1.49## 0.78±0.59*

Bilophila 0.02±0.02 0.00±.0.00# 0.00±0.00##Desulfovibrionaceae

Desulfovibrio 0.09±0.12 0.10±0.07 0.03±0.05*

Helicobacteraceae Flexispira 0.24±0.24 1.09±2.03 0.50±0.47

Proteobacteria

Enterobacteriaceae Proteus 0.00±0.00 0.01±0.01 0.01±0.01Spirochaetes Spirochaetaceae Treponema 4.18±5.25 0.26±0.42# 0.00±0.00##

Tenericutes Mycoplasmataceae Mycoplasma 0.02±0.02 0.04±0.03 0.06±0.05Verrucomicrobia Verrucomicrobiaceae Akkermansia 1.22±2.68 0.00±0.00# 0.00±0.00#

The data are expressed as the mean ± SD (n=6). p vales are calculated with Mann-Whitney U test. (vs. DSS :* P<0.05,**P<0.01;vs. control :# P<0.05,##P<0.01)

Table 2. 1H NMR data of metabolites in rat feces

Number　 Metabolites δ 1H (ppm)1 2-Hydroxyisobutyrate 1.35(s)2 3-Methyl-2-oxovalerate 0.899(t), 1.093(d), 1.438(m), 1.662(m)1.685, 2.928(m), 1.924(s) 3 Alanine 1.484(d), 3.780(q)4 Acetate 1.924(s) 5 Asparagine 2.862(dd), 2.962(m), 3.993(dd)6 Aspartate 2.666(dd), 2.805(dd), 3.898(dd)7 Choline 3.191(s), 3.504(m), 4.085(m)8 Creatine 3.045(s), 3.927(s)9 Dimethylamine 2.719(s)10 Formate 8.460(s)11 Fumarate 6.525(s) 12 Glycerol 3.551(dd), 3.634(m), 3.767(m) 3.76813 Glycine 3.567(s) 14 Histidines 3.144(dd), 3.249(dd), 4.003（dd）, 7.093(s), 7.844(s)15 Isoleucine 0.936(t), 0.993 (d), 1.249(m), 1.452(m), 1.971(m), 3.655(d) 16 Lactate 1.335(d), 4.125(q)17 Leucine 0.925(d), 0.935(s), 1.703(m), 3.724 (d) 18 Methylamine 2.613(s)19 Pyruvate 2.374(s)20 Succinate 2.410(s)21 Threonine 1.335(d), 3.578(d), 4.269(m)22 Xanthine 7.909(s)23 α-Glucose 3.418 (m), 3.547 (m), 3.726(m), 3.742(m), 3.772(m), 3.870 (m), 5.210(d)24 1,3-dihydroxyacetone 3.573(s), 4.42(s)

25 2-Ketoisovalerate 1.127(d), 3.027(m)26 3-Methyl-2-ketovalerate 0.888(t;7.4), 1.102(d;7.0), 1.461(m), 1.727(m), 2.939(m)27 3-hydroxyphenylacetate 3.481(s), 6.757(m), 6.807(d), 6.906(dd), 7.245 (t)28 Adenine 8.196(s), 8.213(s)29 Butyrate 0.899(t), 1.553(m), 2.150(t)30 Cadaverine 1.484(d), 1.727(m), 3.022(t)31 DCA 6.859(d)32 desaminotyrosine 3.817(d)33 Ethanolamine 3.134(d), 7.850(d;7.6)34 Glutarate 1.775(overlap), 2.181(t)35 Hypoxanthine 8.196(s), 8.216(s)36 Imidazole 7.327(s), 8.297(s)37 Isocaproate 0.882(d), 1.449(m), 1.484 (d), 2.189 (t)38 Malonate 3.112(s)39 Methanol 3.366(s)40 Methionine 2.142 (s), 2.169(m), 2.648(t), 3.853(m)41 Methylsuccinate 1.104(d), 2.135 (d), 2.635 (m)42 N-acetyl-glycoprotein 2.03(s)43 NAG 2.042(s), 2.065(s)44 Phenylacetate 3.542(s), 7.317(m), 7.375(m)45 Propionate 1.060(t), 2.187(q), 2.374(s)46 Sarcosine 2.705(s), 3.598(s)47 Taurochenodeoxycholate 0.957, 0.672(s), 0.965(s)48 TCA 0.67(s)49 Trimethylamine 2.871(s)50 Tyrosine 3.068(d), 3.140(dd), 3.941(d), 6.907(m), 7.196 (d/m51 Uracil 5.807(d), 7.542(d) 52 Urocanate 6.40(d), 7.318(d), 7.433(s), 7.885(s)53 Valerate 0.899(t), 1.309(m), 2.178(t)

54 Valine 0.972(d), 1.013(d), 2.275(m), 3.617(d), 7.909(s)55 α-arabinose 3.849(dd), 3.906(m), 3.993(t), 4.023(d), 5.242 (d)56 α-galactose 3.64(dd), 4.08(m), 4.58(d), 5.274 (d)57 α-ketoisocaproate 0.920 (s), 2.054(m), 2.616(d)58 α-ketoisovalerate 1.127(d), 3.022(m)59 α-xylose 3.545(dd), 3.630(m), 3.670 (t), 5.203(d)60 β-arabinose 3.525(dd), 3.670 (t), 3.856(dd), 3.945(m), 4.523(d)61 β-L-Fucose 1.251(d：6.5), 3.452(m), 3.645 (m), 3.804 (m) , 4.561 (d;7.9)62 β-xylose 3.23(dd), 3.46(t), 3.63(m), 3.932(dd), 4.570(d)

Abbreviation: TCA, Taurocholic acid; DCA, Deoxycholic acid; NAG, N-Acetyl group contained glycoprotein

Table 3. 1H NMR data of metabolites in rat urine

1H NMR Data of Metabolites in Rat Urine

Number Metabolites δ 1H (ppm)1 1-Methylnicotinamide 4.476(s),8.181(t),8.894(d),8.960(d),9.272(s)2 2-Hydroxy-3-methylvalerate 0.8(t),0.93(d),1.15(m),1.35(m),1.75(m),3.87(d)3 2PY 6.67(d), 7.97(dd), 8.34(d), 3.65(s)4 3-Hydeoxyphenylacetate 3.490(s),6.794(m),6.872(m or ov),7.258(tr or ov)5 3-Hydroxyisovalerate 1.275(s), 2.357(s)6 4-Hydroxyhippurate 3.978(s),6.976(d),7.765(d)7 4-hydroxyphenylacetate 6.87(d)8 4PY 6.70(d),7.83(dd) ,8.56(d) ,3.90(s)9 4-Pyridoxate 2.429(s),7.834(s)10 Acetamide 1.99(s) 11 Acetoacetate 2.266(s),3.434(s)12 Acetone 2.229(s)13 Acetylcarnitine 2.159(s), 2.504(m), 2.631(m), 3.202(s), 3.621(d), 3.829(dd), 14 Allantoin 5.38(s), 6.02(s), 7.27(s), 8.01(s)15 Betaine 3.273(s),3.927(s)16 Carnitine 2.41(t),3.230(s),3.405(m),4.554(m)17 cis-aconitate 3.123(s/d),5.711(t)18 Creatinine 3.043(s),4.052(s)19 Dimethyl sulfone 3.145(s)20 Galactarate 3.95(s), 4.26(s)21 Galactonate 4.26(s)22 Glycerate 3-phosphate 4.22(m), 4.06(d), 3.84(d)

23 Glycolate 3.932(s)24 Guanidoacetate 3.801(s)25 Guanine 7.69(s)26 Hippurate 3.975(d), 7.553(t), 7.642(t), 7.837(d), 8.508(s）27 Hypotaurine 3.36(t)28 Indole-3-acetate 3.681 (s), 7.161 (t), 7.216 (t), 7.507 (d), 7.635(d)29 Isobutyrate 1.053 (d), 2.382(m)30 Isopropanol 1.146 (d), 4.028(m)31 Isovalerate 0.895(d), 1.929(m), 2.049(d)32 Isovalerylglycine 0.925(d), 1.994(m), 2.166(d),3.746(d)33 Ketoleucine 0.944 (d), 2.100 (m), 2.617(d）

34 Lactose 3.276 (t), 3.507 (m), 3.604 (m), 3.710 (m), 3.768 (m), 3.847 (m), 3.936 (m), 4.455 (d), 4.660 (d), 5.211(d)

35 Methylguanidine 2.828 (s), 3.356(s)36 N, N-dimethylglycine 2.925 (s), 3.712(s)37 N6, N6, N6-trimethyllysine 3.109(s)38 N-acetylglutamate 4.13(t), 1.83(m), 2.27(t), 2.04(s)39 N-Acetylglycine 2.044 (s), 3.763 (d), 7.997(s)40 N-methylhydantoin 2.91(s), 4.08(s)41 N-Nitrosodimethylamine 3.162 (s), 3.802(s)42 N-Phenylacetylglycine 3.67(s), 3.74(d), 7.98(s)43 O-Acetylcholine 2.143 (s), 3.202 (s), 3.711 (t/m), 4.529(m)44 Oxypurinol 8.209(s)45 Pantothenate 0.94(s), 0.90(s), 3.52(s), 3.40(s), 4.00(s), 3.45(t), 2.43(t) 46 p-cresol 2.253 (s), 6.817 (d), 7.130(d)47 p-Cresol glucuronide 2.299 (s), 7.055 (m), 7.237(m)48 p-cresol sulfate 2.347(s), 7.217 (d), 7.293(s,d,d)49 Phenylacetylglycine 3.680 (s), 3.746 (d), 7.365 (t), 7.425(t)

50 Phosphorylcholine 3.203 (s), 3.671 (t), 4.190（dd）51 Propylene glycol 1.14(d), 3.44(dd), 3.54(dd), 3.88(m)52 Succinimide 2.78(s)53 Thiamine 9.42(s), 8.04(s), 5.45(s), 3.89(t), 3.18(t), 2.56(s), 2.49(s)54 Trans-Aconitate 3.435 (s), 6.571（s）55 Trigonelline 4.438 (s), 8.084 (t), 8.837 (t/d), 9.122（s）56 TMAO 3.266(s)57 β-Glucose 3.243 (t), 3.478 (m), 3.492 (m), 3.864 (m), 3.927 (s), 4.660(d)58 4-hydroxyphenylacetate 3.45(s), 6.87(d), 7.16(d)59 3-hydroxyisobutyrate 1.06(d), 2.48(m), 3.52(m), 3.71(m)60 GPC 3.24(s), 3.69(m), 4.33(m)61 NAG 2.04(s) 62 PC 3.22(s), 3.60(m), 4.17(m)63 Dimethylglycine 2.92(s), 3.72(s)64 2-Hydroxyisobutyrate 1.35(s)65 3-Methyl-2-oxovalerate 0.899(t), 1.093(d), 1.438(m), 1.662(m), 2.928(m)66 Alanine 1.489(d), 3.780(q) 67 Acetate 1.927(s)68 Asparagine 2.862(dd), 2.962(m), 3.995(dd)69 Choline 3.191(s), 3.504(t/m), 4.085(m)70 Citrulline 1.558(m), 1.865(m), 3.124(dd), 3.743(t)71 Creatine 3.028/3.047(s), 3.926(s)72 DMA 2.719(s)73 Formate 8.467(s)74 Fumarate 6.524(s)75 Glycine 3.566(s)76 Isoleucine 0.936(t), 0.995(d), 1.249(m), 1.452(m), 1.971(m), 3.655(d)77 Lactate 1.331(d), 4.115(q)

78 Leucine 0.925(d), 0.936(s/d), 1.703(m), 3.725(d)79 Pyruvate 2.373（s）80 p-Hydroxyphenylacetate 3.445(s), 6.865 (d), 7.169(d)81 Succinate 2.413(s)82 Taurine 3.243(t), 3.435(t)83 Threonine 1.330(d), 3.579(d), 4.269(m)84 Xanthine 7.909(s)

Abbreviation: 2PY, N'-methyl-2-pyridone-5-carboxamide; 4PY, N'-methyl-4-pyridone-3-carboxamide; TMAO, Trimethylamine-N-oxide; GPC, Glycerophosphocholine; NAG, N-Acetyl group contained glycoprotein; PC, Phosphorylcholine; DMA, Dimethylamine;

Table 4. Screened metabolites with significant difference in fold change (FC) value in feces samples.

DSS / Control 　 DSS+GA / Control 　 DSS+GA / DSSNumber Metabolites

FC p value Trend 　 FC p value Trend 　 FC p value Trend1 Valerate 2.618 0.007 ↑** 0.450 0.012 ↓*2 Butyrate 1.987 0.014 ↑* 0.532 0.013 ↓*3 Glutarate 1.740 0.01 ↑* 0.578 0.012 ↓*4 Taurochenodeoxycholate 1.655 0.009 ↑**5 Glycine 1.563 0.005 ↑** 1.544 0.002 ↑**6 Methionine 1.517 0.006 ↑** 0.712 0.013 ↓*7 3-Methyl-2-ketovalerate 1.515 0.015 ↑* 0.640 0.013 ↓*8 Propionate 1.509 0.014 ↑* 0.635 0.007 ↓**9 Methylsuccinate 1.503 0.014 ↑* 0.699 0.024 ↓*10 3-Methyl-2-oxovalerate 1.465 0.029 ↑* 0.627 0.013 ↓*11 Acetate 1.465 0.027 ↑* 0.688 0.030 ↓*12 Dimethylamine 0.809 0.002 ↓**13 Adenine 0.800 0.046 ↓*14 Uracil 0.776 0.025 ↓*15 Fumarate 0.739 <0.001 ↓*** 0.733 0.003 ↓**16 Deoxycholic acid 0.637 <0.001 ↓*** 0.703 0.002 ↓**17 Asparagine 1.264 <0.001 ↑***18 Desaminotyrosine 1.261 0.005 ↑** 1.212 0.032 ↓*19 Alpha-arabinose 1.208 0.005 ↑**20 Xanthine 0.750 <0.001 ↓***21 α-galactose 1.234 <0.001 ↑***22 β-xylose 1.280 0.003 ↑**23 β-arabinose 1.249 0.004 ↑**24 α-xylose 1.240 0.008 ↑**

25 Isocaproate 　 　 　 　 　 　 　 　 0.782 0.018 ↓**p<0.05; **p<0.01, ***p<0.001

Table 5. Screened metabolites with significant difference in fold change (FC) value in urine samplesDSS / Control 　 DSS+GA / Control 　 DSS+GA / DSS

Number MetabolitesFC p value Trend 　 FC p value Trend 　 FC p value Trend

1 Oxypurinol 0.337 <0.001 ↓*** 2.771 <0.001 ↑***2 Xanthine 0.588 <0.001 ↓*** 1.691 <0.001 ↑***3 4-Hydroxyhippurate 0.714 <0.001 ↓*** 0.648 <0.001 ↓***4 Hippurate 0.719 <0.001 ↓*** 0.650 <0.001 ↓***5 cis-aconitate 0.792 <0.001 ↓***6 Galactarate 0.815 <0.001 ↓***7 Isovalerate 1.421 <0.001 ↑*** 1.280 <0.001 ↑***8 Acetoacetate 0.723 0.045 ↓*9 Succinate 1.605 0.021 ↑*10 Fumarate 0.806 0.020 ↓*11 Isopropanol 0.707 0.016 ↓* 0.742 0.033 ↓**12 Acetate 2.154 0.011 ↑* 1.316 0.002 ↑**13 Taurine 0.646 0.008 ↓**14 trans-Aconitate 0.506 0.008 ↓**15 Trigonelline 0.830 0.006 ↓** 1.219 0.002 ↑**16 4-Pyridoxate 0.741 0.005 ↓** 0.628 <0.001 ↓***17 2-Hydroxyisobutyrate 1.542 0.003 ↑** 1.338 <0.001 ↑***18 Formate 1.659 0.001 ↑** 0.661 0.010 ↓**19 Indole-3-acetate 0.674 <0.001 ↓***20 succinimide 1.239 <0.001 ↑***21 p-Cresol glucuronide 0.753 <0.001 ↓***22 Glycolate 0.783 <0.001 ↓***23 Asparagine 0.800 <0.001 ↓***24 Phenylacetylglycine 　 　 　 　 0.797 <0.001 ↓*** 　 　 　 　

*p<0.05; **p<0.01, ***p<0.001